Substituted metal-phthalocyanines, their preparation and the use thereof

ABSTRACT

The present invention is directed to metal-phthalocyanines of general formula (I) to the corresponding conjugates, the processes for their preparation and use in the photodynamic therapy of microbial infections (viral, bacterial and mycotic), tumor, pre-cancerous and proliferative pathologies and/or in the diagnosis, as well as for blood and blood derivatives sterilization.

This application is a Divisional of prior application Ser. No.10/311,663 filed Dec. 16, 2002 now U.S. Pat. No. 7,144,879.

FIELD OF THE INVENTION

The present invention refers to metal-phthalocyanines of formula (I)hereafter reported, which are photosensitizer compounds of therapeuticuse, characterised by absorption and fluorescence in the red region ofthe visible spectrum. Said compounds are useful for the treatment anddiagnosis of various infectious diseases and of diseases characterisedby cellular hyperproliferation, in particular tumours, psoriasis,actinic keratosis, atheromas, endoarterial hyperplasia and prostatehyperplasia; said compounds are useful as well for blood and bloodderivatives sterilisation.

STATE OF THE ART

Organic molecules containing the chromofluorophore macrocycle of thephthalocyanine are known to produce reactive derivatives of oxygen, inparticular singlet oxygen or radicals, by interacting with visiblelight.

Compounds having a basic phthalocyanine structure are used in therapy,for example in photodynamic therapy (PDT) and/or for diagnostic purposes(E. Ben-Hur and I. Rosenthal, Int. J. Radiat. Biol., Vol. 47, pp.145-147, 1985).

Other photosensitising agents having applications in photodynamictherapy (PDT) and diagnosis are Zn(II)-phthalocyanines and theconjugates thereof described in the European patent application No. EP98115036 in the name of the Applicant.

Even though the research in this field has made a lot of progress andphotosensitising products defined as “second generation products” havebeen synthesised, their therapeutic application is still limited sincetheir efficacy against pathogenic agents or tumour cells is notsufficiently high or selective.

Up to today, the main therapeutic application of photosensitisingmolecules is associated with their anticancer activity and is based onthe use of porphyrin photosensitising agents (Gomer C. J., Seminars inHematology, Vol. 26, pp. 27-34, 1989), which, albeit giving promisingresults in palliative or curative treatment of different neoplasms, aremarkedly limited by low efficacy and selectivity and have prolongedpersistence in the skin which may cause phenomena of generalisedphotosensitivity (Jori G., J. Photochem. Photobiol., B: Biol., Vol. 36,pp. 87-93, 1996).

The non-optimal distribution of first generation photosensitizers aredue to the poor selectivity, which is related to their physical-chemicalfeatures.

It is therefore evident the importance of developing derivativessuitable for therapeutic and diagnostic applications, as thephthalocyanine compounds described in the present invention.

The main characteristics, which makes phthalocyanine derivativessuitable for therapy and/or diagnostic purposes in vivo, are thefollowing:

i) low dark toxicity, high quantum yield in singlet oxygen productionand/or a high fluorescence quantum yield;

ii) capability of being activated by red or near infrared light,radiations able to penetrate deeply into tissues;

iii) presence of substituents having suitable photodynamic andphysical-chemical features, among which only one substituent bears areactive or potentially activatable functional group, allowing thesite-specific conjugation of the photosensitive molecule tomacromolecular carriers if required.iv) sufficient solubility in water for a good bioavailability, fastmetabolism and a preservation of the biologic properties of theconjugated macromolecular carrier.

In addition to the above mentioned characteristics, it has been recentlydisclosed in scientific literature that the number and charge ofsubstituents effect the in vitro and in vivo phototoxicity of thecompounds (Brasseur et al., Photochem. Photobiol., vol. 45, pp. 581-586,1987; Brasseur et al., Photochem Photobiol., vol. 47, pp. 705-711,1988).

In particular, the highest phototoxicity has been shown when twoadjacent sulphonic groups are present, which has been related to anincreased capacity of penetration into the tumour cells membrane(Brasseur et al., Photochem. Photobiol., vol. 46, pp. 739-744, 1987;Paquette et al., vol. 47, pp. 215-220, 1988; Margaron et al., PhotochemPhotobiol., vol. 62, pp. 217-223, 1996).

It has been moreover proved that the addition of hydrophobic groups tosulphonated phthalocyanines generates an increase of the amphiphilicproperties, a higher cellular uptake and a greater photocytotoxicity(Paquette et al., J. Chim. Phys., vol. 88, pp. 1113-1123, 1991).

SUMMARY OF THE INVENTION

In spite of what reported in the above cited literature about thestudies on structures for photosensitising agents showing photocytotoxicproperties, and from which it comes out that an optimal uptake occurswhen two strong anionic groups are present on adjacent rings of themacrocycle so to form an amphiphilic molecule with a hydrophobic matrix,the Applicant has surprisingly found that phthalocyanines substituted inspecific positions of only one ring of the macrocycle with cationicgroups or with protonable groups, are particularly active, and areparticularly effective in inducing the in vitro photoinactivation.

Object of the present invention, are therefore the metal-phthalocyaninesof general formula (I)

in which:M is chosen in the group consisting of Zn, Si(OR₈)₂, Ge(OR₈)₂ and AlOR₈;R is H or a group selected from alkyl, alkenyl and alkyloxy group,linear or branched, having from 1 to 10 carbon atoms provided that, whenR is different from H, the positions 8, 11, 15, 18, 22, 25 or 9, 10, 16,17, 23, 24 are substituted; andR₁ and R₂, equal or different from one another, are H or a cationicgroup or a protonable group, provided that when R₁ and R₂ are the same,they are not H and are in the positions 1,4 or 2,3, whereas, when onlyone between R₁ and R₂ is different from H, the position 1 or 2 issubstituted, or R₁ and R₂, taken together, form a saturated orunsaturated heterocycle, possibly substituted, which may contain up totwo heteroatoms chosen in the group consisting of N, O and S;R₈ is chosen from between H and C1-C15 alkyl,and their pharmaceutically acceptable salts.

Further object of the present invention are the above formula (I)compounds site-specifically conjugated with bio-organic carriers, suchas aminoacids, polypeptides, proteins and polysaccharides.

Said compounds of formula (I), as well as the corresponding conjugates,are useful for the treatment of microbial infections (viral, bacterialand mycotic), tumour, pre-cancerous and proliferative pathologies inphotodynamic therapy, and are analogously useful as diagnostic agentsfor the identification of pathologically affected areas as well as forthe photodynamic sterilisation of blood and blood derivatives.

Features and advantages of compounds of formula (I) according to thepresent invention will be illustrated in detail in the followingdescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: survival (%) vs. irradiation time (min.) with red light at 100mW/cm² for E. coli 04 previously incubated for 5 min. with 2.5 μM ofcompound 18.

FIG. 2: survival (%) of the colony forming units (CFU) of Candidaalbicans vs. concentration (μM) of the following compounds according tothe invention, in comparison with structurally similar compoundspreviously cited in literature:

—▪— indicates the curve obtained by using the compound 20 prepared asdescribed in Example 3.

—●— indicates the curve obtained by using the compound 19 prepared asdescribed in Example 3.

. . . ∇ . . . shows the curve obtained by using the compound PPC asreported by Minnoch A. et al. J. Photochem. Photobiol. 32: 159-164(1996).

—▾— shows the curve obtained by using the compound identified asCompound 42 in the European Patent Application No. 98115036.0 in thename of the Applicant.

. . . □ . . . shows the curve obtained by using Zn phtalocyaninecommercialised by Aldrich.

. . . ◯ . . . shows the curve obtained by using the compound T₄MPyP asreported by Merchat M. et al. J. Photochem. Photobiol. 32: 153-157(1996).

FIG. 3: shows the variation of CFU for different concentrations ofCandida albicans cells, incubated for 60 min. with 1 μM of the compound19 prepared as described in Example 3, then irradiated with 100 mW/cm²light. On the y-axis cells survival (%) is reported.

DETAILED DESCRIPTION OF THE INVENTION

The present invention makes it possible to meet the above mentionedrequirements thanks to the metal-phthalocyanines of general formula (I).

According to the present invention Zn(II)-phthalocyanines are preferred.

Compounds having formula (I) according to the present invention bear thesame substituents in specific positions on the three benzo-rings of thephthalocyanine nucleus, which are different from the forth one bearingat least one cationic group or a protonable group.

By protonable group according to the above general formula (I) an aminicgroup is preferably meant.

According to a preferred embodiment of the present invention, when onesubstituent between R₁ and R₂ is H, the other one is (X)_(p)R₃, whereinX is chosen in the group consisting of O, S, —NR₆ and —CH₂—; and R₃ is

where:Y is chosen in the group consisting of C1-10 alkyl and phenyl, possiblysubstituted, or it forms with the Z group, to which it is bound, asaturated or unsaturated heterocycle, possibly substituted, which maycontain up to two heteroatoms chosen in the group consisting of N, O andS;Z is chosen in the group consisting of —N, —CH₂N and —CONHCH₂CH₂N;R₄ and R₅, equal or different from one another, are chosen in the groupconsisting of C1-15 alkyl and phenyl, or form with the Z group, to whichthey are bound, a saturated or unsaturated heterocycle, possiblysubstituted, which may contain up to two heteroatoms chosen in the groupconsisting of N, O and S;R₆ and R₇, equal or different from one another, are chosen in the groupconsisting of H and C1-C15 alkyl;m, n, p, w, t and u, independently from one another, are 0 or 1; andv is an integer comprised between 1 and 3.

By saturated or unsaturated heterocycle possibly substituted, as definedin the above general formula, the following are preferably meant:morpholine, piperidine, pyridine, pyrimidine, piperazine, pyrrolidine,pyrroline, imidazole, aniline, and julolidine (2,3,6,7-tetrahydro-1H,5Hbenzo[ij]quinolizine).

According to the invention, the preferred products are those in whichthe group (X)_(p)R₃ contains substituents bearing tertiary or quaternarynitrogen.

In particular, the said group (X)_(p)R₃ is preferably represented by:

The present compounds show valuable photodynamic characteristics makingthem useful in photodynamic therapy (PDT) against bacterial, fungal andviral infections, for various hyperproliferative diseases as well as forphotosterilization of blood and blood derivatives such as platelets anderythrocytes. In this particular case the present compounds can be addeddirectly, or previously bound to suitable matrix, to blood or bloodderivatives, according to the known techniques and thereafterirradiated. Moreover they can be used as diagnostic agents for theidentification of pathologically affected areas.

The present products possess a molar absorption coefficient higher thanthe photosensitising agents presently used in therapy, which representsan important requirement for an effective therapeutic response.

These products may be activated by tissue penetrating radiation having awavelength longer than 650 nm, and hence are suitable for the PDTagainst diseases, both dermatological and internal.

The products formed by photobleaching of those compounds are non toxic.This finding reinforces their usefulness as therapeutics since afterhaving exploited their action the compounds are inactivated by the lightand then are no more potentially toxic in vivo.

The present compounds are active in the singlet oxygen production orallow the production of reactive species of oxygen under conditions ofpoor oxygenation. Such requirement is particularly important because itallows to treat specifically anaerobic microorganisms or tumour cells,well-known characterised by an environment poor of oxygen.

In particular, the exemplified products possess very high efficiency formicro-organisms such as yeast fungi and mycoplasma, Gram-positive andGram-negative bacteria, showing the capability of specific localisationon micro-organisms compared to the mammalian host cells.

The finding about differential toxicity between host cells andmicro-organisms strengthen the importance of the claimed products (I).

The present invention comprises also the above described formula (I)compounds site-specifically conjugated with a bio-organic carrier ableto direct the molecule to a definite target.

The carrier is usually chosen among molecules having well-known specificbinding capacities, for example aminoacids (preferably basicaminoacids), polypeptides, (preferably consisting of basic aminoacids),proteins and polysaccharides normally used for targeting purposes.

The binding phthalocyanine(I)/carrier may occur for example between therelated amino or carboxyl group, or may occur involving other specificfunctional groups on the phthalocyanine moiety or on the carriermolecule.

Functional groups such as thiol, maleimide derivatives, α-bromo estersand amides, diazonium salts and azido derivatives can be introducedfollowing current to procedures in order to pre-functionalise both thephthalocyanine or the carrier depending upon the selected carrier itselfand its stability.

The compounds of the present invention can be prepared, by condensationin the homogeneous as well as in the heterogeneous phase ofphthalonitriles properly substituted, according to reaction schemesknown in organic chemistry.

For example, the amino substituted Zn(II)-phthalocyanines of formula (I)can be prepared according to the herein described processes.

a) Liquid phase by mixed condensation method using two different1,2-benzenedicarbodinitriles, having suitable substituents, defined bythe following formula (II) and formula (III)

wherein R, R₁ and R₂ are as defined above. In the formula (II) compound,when R₁ and R₂ are the same, the positions 3,6 or 4,5 are substituted,whereas, when one substituent between R₁ and R₂ is H, the other one isin the position 3 or 4. In the formula (III) compound, when R isdifferent from H, the positions 3,6 or 4,5 are substituted.

The phthalonitriles of formula (II) and (III) are mixed in differentratios (1:1 to 1:6) by using neat 1,8-diazabicyclo-[5.4.0]-undec-7-ene(DBU), or with solvent (dichloromethane, methanol,N,N-dimethylformamide), in the presence of anhydrous zinc(II) acetate atvarious temperatures and reaction times, to afford a mixture ofcompounds. The mixture is first purified by extensive washing steps withwater and organic solvents (i.e. precipitation DMF/water, extractionswater/organic solvent) and then by chromatography (i.e. by using silicagel, deactivated basic or neutral aluminium oxide, Sephadex) followed byfurther washings of the separated products with organic solvents (Et₂O,AcOEt, CH₂Cl₂, CHCl₃, acetone, methanol).

b) Solid Phase

Some of the phthalocyanines of formula (I), having suitablesubstituents, can also be prepared by solid phase synthesis with the aimat avoiding the time-consuming difficult purification proceduresforeseen by the statistical synthesis. The preparation process, startingfrom a dinitrile bound to a solid phase reacted with a differentlysubstituted dinitrile previously transformed into diaminoisoindolylderivative, has already been disclosed [Tetrahedron Letters, vol. 23(30) pp. 3023-3026 (1982); J. Org. Chem., vol. 56, pp. 82-90 (1991)];however, the described procedure is a refinement of the above cited oneand specifically leads to amino substituted metal phthalocyanines neverreported so far.

Cationic metal phthalocyanines of formula (I) can be prepared byreacting the corresponding neutral compounds obtained as described abovewith an excess of neat alkyl iodide, or in the presence of a suitablesolvent (i.e. N-Methyl-2-pyrrolidinone, DMF, methanol, chloroform), attemperature comprised between room temperature and reflux for a reactiontime comprised between 1 hour and 10 days). The crude products can begenerally purified by several washings with various organic solvents,such as Et₂O, CH₂Cl₂, and CHCl₃, ethyl acetate or acetone.

Each prepared compound can be identified by means of variousspectroscopic techniques as ¹H-NMR and ¹³C-NMR, MS andspectrophotometrically characterised in the UV-Vis region.

Hereinafter the preparation of specific compounds of formula (I) isreported for better illustrating the invention.

EXAMPLE 1 Synthesis of the Compound of Formula (I) in which M is Zn,R=R₂=H and R₁=1,3-bis-(dimethylamino)-2-propyloxy in the Position 2(Compound 1)

0.272 g of 4-[1,3-bis-(dimethylamino)-2-propyloxy]-1,2-benzenedicarbodinitrile (1 mmol) and 0.384 g of 1,2-benzenedicarbodinitrile (3mmol) are dissolved in a little amount of methanol and added of Zn(OAc)₂(0.176 g; 0.96 mmol) and DBU (0.66 ml; 0.42 mmol). The mixture is heatedat 150° C., under an inert atmosphere, for 3 h and 30 min. The bluemixture is dissolved in DMF and precipitated with basic water severaltimes, then is purified by flash-chromatography on silica gel, elutingwith Et₂O/DMF (4:1), EtOAc/DMF (4:1), EtOAc/DMF (1:1), EtOAc/DMF (1:2),DMF. The product of interest is further purified by Washing with Et₂Oand acetone. Blue-violet powder.

UV-vis (DMF) λ_(max) (ε, M⁻¹cm⁻¹) 344, 606, 672 (2.7635×10⁵).

¹H NMR (DMSO-d₆) δ ppm 9.42-9.30 (m, 6H), 9.23 (d, 1H), 8.95 (s, 1H),8.30-8.20 (m, 6H), 7.90-7.80 (m, 1H), 5.25-5.18 (m, 1H), 2.90 (d, 4H),2.49 (s, 12H).

ESI-MS, m/z

Using the above described procedure, the following products wereobtained:

Compound 2: compound of formula (I) in which M is Zn, R=R₂=H andR₁=pyridin-4-yl-oxy in the position 2; blue-violet powder; UV-vis (DMF)λ_(max), nm (ε, M⁻¹cm⁻¹) 350, 606, 674 (8.9670×10⁴). ¹H NMR (DMSO-d₆) δppm 9.40-8.95 (m, 7H), 8.62 (d, J=7.50 Hz, 2H), 8.35-8.10 (m, 8H), 6.59(d, J=7.43 Hz, 2H). ESI-MS, m/z 670 [M+H]⁺.

Compound 3: compound of formula (I) in which M is Zn, R=R₂=H andR₁=3-(dimethylamino)phenoxy in the position 2; blue-violet powder.UV-vis (DMF) λ_(max), nm 346, 605, 671. ¹H NMR (DMSOd₆) δ ppm 9.48-9.05(m, 7H), 8.72-8.65 (m, 1H), 8.32-8.12 (m, 6H), 7.90-7.80 (m, 1H),7.52-7.38 (m, 1H), 6.88-6.75 (m, 3H), 3.08 (s, 6H). ESI-MS, m/z 712.3[M⁺H]⁺.

Compound 4: compound of formula (I) in which M is Zn, R=R₂=H andR₁=1-methylpiperidin-4-yl-oxy in the position 2; blue-violet powder.UV-vis (DMF) λ_(max), nm 343, 606, 673. ¹H NMR (DMSO-d₆) δ ppm 9.45-9.30(m, 5H), 9.17 (d, J=8.51 Hz, 1H), 8.794 (d, J=1.7 Hz, 1H), 8.33-8.18 (m,7H), 7.775 (dd, J=8.21 Hz, J=2.1 Hz, 1H), 5.20-4.98 (m, 1H), 2.95-2.80(m, 2H), 2.60-2.40 (m, 2H), 2.40-2.12 (m, 2H; s, 3H), 2.15-1.94 (m, 2H).ESI-MS, m/z 690.2 [M⁺H]⁺.

Compound 5: compound of formula (I) in which M is Zn, R=H andR₁=R₂=pyridin-4-yl-oxy in the positions 2,3; blue-violet powder. UV-vis(DMF) λ_(max), nm 347, 666, 684. ¹H NMR (DMSO-d₆) δ ppm 9.43-9.39 (m,6H), 8.30-8.22 (m, 8H), 8.12 (d, J=7.6 Hz, 4H), 6.39 (d, J=7.6 Hz, 4H).

Compound 6: compound of formula (I) in which M is Zn, R=R₂=H andR₁=1,3-bis-(dimethylamino)-2-propyloxy in the position 1; blue-violetpowder. UV-vis (DMF) λ_(max) nm 336, 611, 677. ¹H NMR (DMSO-d₆) δ ppm9.50-9.42 (m, 6H), 9.07 (bd, J=7.33 Hz, 1H), 8.40-8.12 (m, 7H), 7.91(bd, 7.95, 1H), 5.50-5.38 (m, 1H), 3.25-3.17 (m, 4H), 2.47 (s, 12H).

Compound 7: compound of formula (I) in which M is Zn, R=H, andR₁=R₂=3-(piperidin-1-yl)propyloxy in the positions 1,4; blue-greenpowder. UV-vis (DMF) λ_(max), nm 335, 622, 690. ¹H NMR (DMSO-d₆) δ ppm9.45-9.35 (m, 6H), 8.27-8.18 (m, 6H), 7.72 (s, 2H), 4.76-4.71 (m, 4H),3.10-3.03 (m, 4H), 2.57-2.51 (m, 12H), 1.63-1.36 (m, 12H). FAB-MS, m/z861 [M⁺H]⁺.

Compound 8: compound of formula (I) in which M is Zn, R=H andR₁=R₂=3-(dimethylamino)phenoxy in the positions 2,3; blue-violet powder.UV-vis (DMF) λ_(max) 344, 606, 672. ¹H NMR (DMSO-d₆) δ ppm 9.39-9.28 (m,6H), 8.95 (s, 2H), 8.20-8.15 (m, 6H), 7.45-7.35 (m, 4H), 6.72-6.73 (m,4H), 3.03 (s, 12H).

Compound 9: compound of formula (I) in which M is Zn, R=R₂=H andR₁=pyridin-2-yl-oxy in the position 2; blue-violet powder. UV-vis (DMF)λ_(max), nm 343, 606, 672. ¹H NMR (DMSO-d₆) δ ppm 9.38-9.11 (m 7H),8.50-8.40 (m, 1H), 8.32-8.07 (m, 9H), 7.88-7.75 (m, 1H), 6.85-4.68 (m,1H).

Compound 10: compound of formula (I) in which M is Zn, R=R₂=H andR₁=pyridin-3-yl-oxy in the position 2; blue-violet powder. UV-vis (DMF)λ_(max), nm 343, 606, 672. ¹H NMR (DMSO-d₆) δ ppm 9.40-8.85 (m, 8H),8.72-8.62 (m, 1H), 8.34-8.10 (m, 7H), 8.09-7.95 (m, 1H), 7.94-7.68 (m,2H).

Compound 11: compound of formula (I) in which M is Zn, R=R₂=H andR₁=3-(dimethylamino)phenoxy in the position 1; blue-violet powder.UV-vis (DMF) λ_(max), nm 333, 607, 674. ¹H NMR (DMSO-d₆) δ ppm 9.48-9.28(m, 6H), 9.23 (d, J=7.46 Hz, 1H), 9.99 (d, J=7.26 Hz, 1H), 8.38-8.11 (m,7H), 7.81 (d, J=7.65 Hz, 1H), 7.14 (dd, J₁=J₂=8.40 Hz, 1H), 7.10-7.00(m, 1H), 6.53 (bd, J=8.40 Hz, 1H).

Compound 12: compound of formula (I) in which M is Zn, R=H andR₁=R₂=2-(diethylamino)ethylthio in the positions 2,3; blue-violetpowder. UV-vis (DMF) λ_(max) 347, 612, 680. ¹H NMR (DMSO-d₆) δ ppm9.28-9.17 (m, 6H), 8.85 (s, 2H), 8.26-8.15 (m, 6H), 3.59-3.52 (bt, 4H),3.07-3.00 (bt, 4H), 2.79 (q, J=7 Hz, 8H), 1.20 (t, J=7 Hz, 12H).

Compound 13: compound of formula (I) in which M is Zn, R=H andR₁=R₂=pyridin-3-yl-oxy in the positions 2,3; blue-violet powder. UV-vis(DMF) λ_(max) 346, 605, 671. ¹H NMR (DMSO-d₆) δ ppm 9.10-9.32 (m, 6H),8.77 (bs, 2H), 8.55 (d, J₁=4.6 Hz, 2H), 8.25-8.18 (m, 8H), 7.87 (bd,J₂=8.3, 2H), 7.61 (dd, J₁=4.6 Hz, J₂=8.3, 2H).

Compound 14: compound of formula (I) in which M is Zn, R=R₂=H andR₁=2-(dimethylamino)ethyloxy in the position 1; blue-violet powder.UV-vis (DMF) λ_(max) 334, 611, 677. ¹H NMR (DMSO-d₆) δ ppm 9.36 (m, 8H),8.95 (d, J=7.5 Hz, 1H), 8.23 (m, 7H), 8.12 (dd, J₁=7.7 Hz, J₂=7.5 Hz,1H), 7.75 (d, J=7.7 Hz, 1H), 4.83 (bt, 2H), 2.62 (s, 6H).

Compound 15: compound of formula (I) in which M is Zn, R=R₂=H andR₁=2-(piperidin-1-yl)ethyloxy in the position 1; blue-violet powder.UV-vis (DMF) λ_(max) 337, 611, 677. ¹H NMR (DMSO-d₆) δ ppm 9.30-9.27 (m,8H), 8.83 (d, J=7.6 Hz, 1H), 8.22-8.19 (m, 7H), 8.04 (dd, J₁=J₂=7.6 Hz,1H), 7.66 (d, J=7.6 Hz, 1H), 4.82 (bt, 2H), 2.88 (bt, 2H), 2.52 (m, 4H),1.64-1.47 (m, 6H).

Compound 16: compound of formula (I) in which M is Zn, R=R₂=H andR₁=2-(piperidin-1-yl)ethyloxy in the position 2; blue-violet powder.UV-vis (DMF) λ_(max) 347, 607, 671. ¹H NMR (DMSO-d₆) δ ppm 9.30-9.23 (m,8H), 8.99 (D, J=8.4, 1H), 8.61 (bs, 1H), 8.25-8.17 (m, 7H), 7.63 (d,J=8.4 Hz, 1H), 4.61 (bt, 2H), 3.03 (bt, 2H) 2.70 (m, 4H), 1.68-1.53 (m,6H).

EXAMPLE 2 Synthesis of the Compound of Formula (I) in which M is Zn,R=R₂=H and R₁=N-(2-aminoethyl)benzamidoyl-4-oxy trifluoro acetate(Compound 17)

a) Functionalization of the Polystyrene-Based Resin with thePhthalodinitrile

159 mg (0.078 mmol) of diaminoethane-trityl resin (0.49 mmol/g) areswollen in 12.5 mL of DMF. To this suspension 282 mg (0.78 mmol) of thesuccinimide ester of the 4(3′,4′-dicyano)phenoxybenzoic acid are added,and the product is kept under stirring at room temperature for 18 hours.The liquid phase is removed from the resin by vacuum filtration, and theresin is washed several times with small volumes of DMF, CH₂Cl₂ andMeOH.

b) Solid-Phase Condensation Reaction

100 mg (0.78 mmol) of 1,2-dicyanobenzene are dissolved in 2 ml of DMF,the obtained functionalised resin (0.078 mmol) is added to this solutionand the mixture warmed for one hour at 50° C. Then 79 mg (0.43 mmol) ofzinc(II)acetate and 0.322 ml (2.15 mmol) of DBU are added and thesuspension is heated up to 160° C. for 4 hours, under stirring andnitrogen atmosphere. After cooling at room temperature, the two phasesare separated by vacuum filtration, and the solid phase is washed withMeOH and DMF.

c) Separation of the Zn-Phthalocyanine from the Resin

The green-blue resin is suspended in a solution of trifluoroacetic acid(TFA) (375 ml, 5%) and tri-isopropyl silane (TIS) (375 ml, 5%) in CH₂Cl₂(7.5 ml) and kept in this solution for 1.5 hours. The two phases arethen separated by vacuum filtration and the resin is washed with CH₂Cl₂and with small volumes of DMF and MeOH alternatively until the solutionis colourless. The filtrate is concentrated and the blue-green residuepurified by column chromatography and by washing with solvents to give 8mg of the desired product2-7[N-(2-aminoethyl)benzamidoyl-4-oxy]zinc(II)phthalocyanine trifluoroacetate (Compound 17); green-blue solid; UV-vis (DMF) λ_(max), nm;¹H-NMR (DMSOd₆), δ ppm 9.6-9.3 (m, 7H), 9.0-8.75 (s, 1H), 8.75-8.6 (m,1H, disappeared with D₂O), 8.35-8.2 (m, 6H), 8.2-7.9 (m, 6H, modifiedwith D₂O), 7.6-7.4 (m, 2H), 3.7-3.5 (m, 2H), 3.2-3.0 (m, 2H), ESI-MS,m/z: 755 [M⁺H]⁺.

EXAMPLE 3 Synthesis of the Compound of Formula (I) in which M is Zn,R=R₂=H and R₁=1,3-bis-(trimethylammonium)-2-propyloxy diiodide in thePosition 2 (Compound 18)

10 mg of Compound 1 prepared as described above in Example 1, (0.014mmol) is dissolved in 2.5 ml of N-Methyl-2-pyrrolidinone and treatedwith an excess of MeI, leaving the reaction mixture at r.t. for 15 h.The product is precipitated with Et₂O from the mixture, recovered byfiltration and purified by several washings of the precipitate withorganic solvents, thus obtaining the desired product2[1,3-bis-(trimethylammonium)-2-propyloxy]zinc(II)phtalocyaninediiodide; blue powder. UV-vis (DMF) λ_(max) (ε, M⁻¹cm⁻¹) 343, 607, 672(1.9275×10⁵). ¹H NMR (DMSO-d₆) δ ppm 9.55-9.40 (m, 7H), 9.23 (s, 1H),8.42-8.35 (m, 6H), 8.25-8.15 (m, 1H), 6.30-6.10 (m, 1H), 4.45-4.10 (m,4H), 3.55 (s, 18H). ESI-MS, m/z 375.3 [M−2I]²⁺.

Using the above described procedure, the following products were alsoobtained:

Compound 19: compound of formula (I) in which M is Zn, R=R₂=H andR₁=3-(trimethylammonium)phenoxy iodide in the position 2; UV-vis (DMF)λ_(max), nm (ε, M⁻¹ cm⁻¹) 345, 606, 671 (1.7073×10⁵). ¹H NMR (DMSO-d₆) δppm 9.55-9.42 (m, 5H), 9.42-9.35 (m, 1H), 9.05-8.97 (m, 1H), 8.38-8.20(m, 8H), 8.05-7.80 (m, 3H), 7.68-7.60 (m, 1H), 3.77 (s, 9H). ESI-MS, m/z726.4 [M−I]⁺

Compound 20: compound of formula (I) in which M is Zn, R=R₂=H andR₁=1,1-dimethylpiperidinium-4-yl-oxy iodide in the position 2. UV-vis(DMF) λ_(max), nm (ε, M⁻¹cm⁻¹) 341, 606, 671 (1.8197×10⁵). ¹H NMR(DMSO-d₆) δ ppm 9.52-9.39 (m, 5H), 9.35 (d, 1H), 9.00 (d, 1H), 8.40-8.25(m, 7H), 7.95 (dd, 1H).2. ESI-MS, m/z 704.3 [M−I]⁺

Compound 21: compound of formula (I) in which M is Zn, R=H andR₁=R₂=3-(1-methylpiperidinium-1-yl)propyloxy iodide in the positions1,4; blue-green powder. UV-vis (DMF) λ_(max), nm 337, 619, 687. ¹H NMR(DMSO-d₆) δ ppm 9.48-9.39 (m, 6H), 8.31-8.27 (m, 6H), 7.89 (s, 2H),5.10-4.90 (m, 4H), 4.13-4.03 (m, 4H), 3.55-3.45 (m, 8H), 3.23 (s, 6H),2.90-2.65 (m, 4H), 1.90-1.72 (m, 8H), 1.68-1.32 (m, 4H). ESI-MS, m/z1015.4 [M−I]⁺, 444.6 [M−2I]²⁺.

Compound 22: compound of formula (I) in which M is Zn, R H andR₁=R₂=3-(trimethylammonium)phenoxy iodide in the positions 2,3; bluepowder. UV-vis (DMF) λ_(max), nm 342, 606, 672. ¹H NMR (DMSO-d₆) δ ppm9.52-9.38 (m, 6H), 9.30 (s, 2H), 8.45-8.30 (m, 6H), 8.12 (bs, 2H),7.92-7.80 (m, 4H), 7.63-7.58 (bd, 2H), 3.73 (18H).

Compound 23: compound of formula (I) in which M is Zn, R=R₂=H andR₁=1,3-bis-(trimethylammonium)-2-propyloxy diiodide in the position 1;green powder. UV-vis (DMF) λ_(max), nm 334, 609, 676. ¹H NMR (DMSO-d₆) δppm 9.60-9.38 (m, 6H), 9.36-9.30 (m, 1H), 8.52-8.20 (m, 7H), 7.95-7.85(m, 1H), 6.40-6.30 (m, 1H), 4.68-4.38 (m, 4H), 3.44 (s, 18H). ESI-MS,m/z 1005 [M+H]⁺.

Compound 24: compound of formula (I) in which M is Zn, R=R₂=H andR₁=3-(trimethylammonium)phenoxy iodide in the position 1; blue-violetpowder. UV-vis (DMF) λ_(max), nm 333, 607, 674. ¹H NMR (DMSO-d₆) δ ppm9.50-9.30 (m, 6H), 8.78-8.70 (m, 1H), 8.52 (bs, 1H), 8.4-8.06 (m, 7H),8.02-7.92 (m, 1H), 7.72-7.65 (m, 1H), 7.60-7.50 (m, 1H), 7.26-7.34 (m,1H), 3.80 (s, 9H). ESI-MS, m/z 726.3 [M−I]⁺.

Compound 25: compound of formula (I) in which M is Zn, R=H andR₁=R₂=2-(diethylmethylammonium)ethylthio iodide in the positions 2,3;blue-violet powder. UV-vis (DMF), λ_(max), nm 347, 611, 681. ¹H NMR(DMSO-d₆) δ ppm 9.49-9.45 (m, 8H), 8.36-8.31 (m, 6H), 4.11-4.05 (m, 4H),3.85-3.69 (m, 4H), 3.55-3.65 (bq, 8H), 3.25 (s, 6H), 1.42 (t, J=7 Hz,12H).

Compound 26: compound of formula (I) in which M is Zn, R=R₂=H andR₁=1-methylpyridinium-4-yl-oxy in the position 2; blue-violet powder,UV-vis (DMF) λ_(max), nm (ε, M⁻¹cm⁻¹) 350, 606, 674. ¹H NMR (DMSO-d₆) δppm 9.85-9.68 (bd, 2H), 9.42 (bs, 1H), 9.41-9.00 (m, 7H), 8.52-8.41 (bd,1H), 8.40-8.02 (m, 8H). ESI-MS, m/z 684.2 [M−I]⁺.

EXAMPLE 4 Preparation of the Conjugate Between Compound 17 and BovineSerum Albumin (BSA)

Bovine serum albumin (BSA) has been prepared as a 5 mg/ml solutionconcentration in PBS (pH 8.5); the Compound 17, obtained as describedabove in Example 2, has been prepared as DMSO solution (5 mg/ml).

In one experiment, 12.5 equivalents of compound 17 are mixed with 200 μlof BSA solution; the blue suspension is maintained under gentle stirringat 4° C. then 12.5 equiv. of disuccinimidylsuberate (DSS, Pierce) areslowly added and the temperature raised to room temperature in 90minutes. After centrifugation, the BSA-compound 17 conjugation productis purified by gel filtration (Sephadex G25) eluting with PBS (pH 7.2),collecting fractions having a volume of ca. 1 ml.

The conjugation product, visible due to its green-blue colour, isobtained from the second fraction to the fourth one. The labelling ratiohas been determined spectrophotometrically measuring the proteinconcentration and the number of moles of the compound 17 per mole ofBSA.

In the practiced experimental conditions the labelling ratio resultedcomprised between 4 and 5 and may be adjusted by variation of theinitial reagents ratio, according to the needs.

Analogously, conjugates of the other formula (I) compounds according tothe invention can be prepared.

Pharmaceutical Formulations

Therapeutic compositions containing the compounds of the presentinvention include solutions also for the administration by parenteralinjection route, preparations for topical application, etc.

The topical formulations according to the invention are for examplelotions, creams, ointments or gels. Particularly preferred are DMSO orAzone aqueous solutions, up to 50%.

The formula (I) compounds of the present invention having lipophiliccharacteristics may be incorporated in liposomes or microcapsules andused in this form for both types of application mentioned above.

The dosages normally range from 0.1 to 20 mg of compound of formula (I)per kilogram of body weight, preferably 0.2-5 mg/Kg of body weight.

Biocidal Activity

The compounds disclosed in the present invention for the applicationsaccording to PDT show many advantages, such as the very low toxicity inthe absence of light.

Each molecule can be repeatedly excited, with the consequent productionof singlet oxygen or other reactive species by a continuous process.

Due to their short lifetime, they hit the target cell withoutpossibility of diffusion to vicinal cells: singlet oxygen is producedonly in the pathologic site, and the portion that does not react withthe biological target undergoes a rapid decay.

These characteristics are further improved by the specific localisationof the photosensitising agent, by the nature of the photosensitizeritself or guaranteed by a suitable carrier. The photodynamic therapythat uses the present compounds is therefore selective and does notallow for systemic or dermal phototoxicity.

The production of singlet oxygen occurs only simultaneously withirradiation and stops immediately as soon as the irradiation isinterrupted.

The light sources suitable for carrying out the PDT are well known inthe art and comprise properly filtered non coherent white light or laserlight having the required specific wavelength preferably ranging from650 to 750 nm.

The total amount of radiation applied varies according to the treatmentand the localisation of the tissues to be treated.

The amount of radiation ranges usually from 50 to 1000 J/cm², andpreferably from 100 to 350 J/cm².

Antifungal and Antibacterial (Gram-Positive E Gram-Negative) Activity

The compounds synthesized have been assayed for their antifungal andantibacterial (Gram-positive e Gram-negative) activity. For theexperiments the following micro-organisms were used: Candida albicans(ATCC10231; yeast), Staphylococcus aureus (ATCC 6538P, MRSA;Gram-positive), Escherichia coli (04; Gram-negative). All themicro-organisms were used in the experiments in a stationary state ofgrowth. An example of experimental protocol used to assess thephotoinactivation of micro-organisms is described as follows.

Cells in a stationary state of growth have been washed in physiologicalbuffered saline solution (PBS) and diluted in order to obtain a cellsuspension in the range 10⁶÷10⁹ cells/ml in PBS or appropriate medium.

Cell suspensions have then been treated with scalar aliquots of thephotosensitising agent to be tested in the range 30÷0.01 μM, eventuallyas a photosensitizer conjugate. Cells have been Incubated in the dark at37° C. for 1 hour, eventually washed at the end with PBS then irradiatedwith red light (700±30 nm; 10÷100 mW/cm²; 1÷30 minutes). In order todetermine the percentage of cell population after photoinactivation foreach photosensitizer concentration, serially diluted samples resultingfrom the irradiation have been plated on Sabouraud Dextrose Agar (C.Albicans), on Tryptic Soy Agar (S. aureus), or on other suitable culturemedia and the data were compared with dark controls.

Examples of microbial photoinactivation by using some compounds of thepresent invention are given in the figures.

FIG. 1 shows the variation of the percentage survival as a function ofirradiation time (administered light dose) for E. coli 04 previouslyincubated for 5 min. with 2.5 μM of compound 18 prepared as described inExample 3, then irradiated with red light at 100 mW/cm².

FIG. 2 accounts for the decrease of the colony forming units (CFU) ofCandida albicans after treatment with different concentrations of somecompounds described in this invention, in comparison with structurallysimilar compounds previously cited in literature.

Reported in FIG. 3 is the variation of CFU as a function of administeredlight dose for various concentrations of Candida albicans cells,incubated for 60 min with 1 μM of the compound 19 then irradiated with100 mW/cm² red light.

Finally we report in the following Table 1 the survival of 3T3 cells asa model of mammalian cells as well as on some micro-organisms afterphotodynamic treatment with the compound 18 prepared as described inExample 3 in PBS+5% DMSO, as an example to demonstrate the selectivityand efficacy of the described compounds. The irradiation is carried outwith red light (650-750 nm), 50 mW/cm².

TABLE 1 Survival (%) Cells or Irradiation 1 min. Irradiation 5 min.micro-organisms 1 μM 10 μM 1 μM 10 μM *3T3 100 100 97 94 *S. aureus(MRSA) 0.0002 0.0001 0.0001 0.0001 *E. coli 100 100 100 2.34 **C.albicans 0.0001 N.D. 0.0001 N.D. *5 min. of pre-incubation **10 min. ofpre-incubation

Evidence about selectivity is also supported by experiments on thehaemolytic properties of the above described compounds. In the followingTable 2 it is shown that several compounds are not toxic for theerythrocytes as well: in fact no haemolysis is detected after treatmentand irradiation by using photosensitzer concentrations well above theones needed for the complete inactivation of micro-organisms. Thesefindings strengthen the usefulness of the described compounds and enablethem to be used even for the blood and blood derivatives sterlisation.

TABLE 2 % Haemolysis (Light treatment) Concen- 30 J/cm² tration %Haemolysis (50 mW/cm² × Compound (μM) (Dark treatment) 10 min)  4 5 1.51.5 1 1.5 1.2 0.5 4.7 0.9 19 5 7.6 0.6 1 5.0 2.0 0.5 0.8 0.6 0.05 4.00.5 26 1 6.3 5.0 0.5 1.2 0.6 Compound 42 0.5 2.8 3.2 disclosed in EP-A-0.1 1.1 2.0 No. 98115036.0 0.05 0.9 0.6

1. A therapeutic method for the treatment of fungal or bacterialpathological conditions, which comprises administering to a subject in aneed of such a treatment a therapeutically effective amount of apharmaceutical composition containing as active ingredient a compound ofgeneral formula (I)

or a conjugate of a compound of general formula (I) with a macromoleculeselected from the group consisting of aminoacids, polypeptides, proteinsand polysaccharides, optionally in combination with pharmaceuticallyacceptable excipients, in which general formula (I): M is selected fromthe group consisting of Zn, Si(OR₈)₂, Ge(OR₈)₂ and AlOR₈; R is H or agroup selected from alkyl, alkenyl and alkyloxy group, linear orbranched, having from 1 to 10 carbon atoms provided that, when R isdifferent from H, the positions 8, 11, 15, 18, 22, 25 or 9, 10, 16, 17,23, 24 are substituted; and R₁ and R₂, equal or different from oneanother, are H or a group (X)_(p)R₃, wherein X is selected from thegroup consisting of O, S, —NR₆ and —CH₂—; and R₃ is

where: Y is selected from the group consisting of C1-C10 alkyl andphenyl, optionally substituted, or it forms with the Z group, to whichit is bound, a saturated or unsaturated heterocycle, optionallysubstituted, which may contain up to two heteroatoms selected from thegroup consisting of N, O and S; Z is selected from the group consistingof —N, —CH₂N and —CONHCH₂CH₂N; R₄ and R₅, equal or different from oneanother, are selected from the group consisting of C1-C15 alkyl andphenyl, or form with the Z group, to which they are bound, a saturatedor unsaturated heterocycle, optionally substituted, which may contain upto two heteroatoms selected from the group consisting of N, O and S; R₆and R₇, equal or different from one another, are selected from the groupconsisting of H and C1-C15 alkyl; m, n, p, w, t and u, independentlyfrom one another, are 0 or 1; and v is an integer between 1 and 3,provided that when R₁ and R₂ are the same, they are not H and are in thepositions 1,4 or 2,3, whereas, when only one between R₁ and R₂ isdifferent from H, the position 1 or 2 is substituted, or R₁ and R₂,taken together, form a saturated or unsaturated heterocycle, possiblysubstituted, which may contain up to two heteroatoms selected from thegroup consisting of N, O and S; R₈ is selected from H and C1-C15 alkyl.2. The therapeutic method according to claim 1, wherein M is Zn.
 3. Thetherapeutic method according to claim 1, wherein said saturated orunsaturated heterocycle is selected from the group consisting ofmorpholine, piperidine, pyridine, pyrimidine, piperazine, pyrrolidine,pyrroline, imidazole, aniline and julolidine.
 4. The therapeutic methodaccording to claim 1, wherein the said group (X)_(p)R₃ containssubstituents bearing tertiary or quaternary nitrogen.
 5. The therapeuticmethod according to claim 4, wherein the said group (X)_(p)R₃ isselected from the group consisting of:


6. The therapeutic method according to claim 1, wherein in saidcompounds of general formula (I): R=R₂=H, andR₁=1,3-bis-(dimethylamino)2-propyloxy in position 2 (Compound 1); R==H,and R₁=pyridin-4-yl-oxy in position 2 (Compound 2); R==H, andR₁=3-(dimethylamino)phenoxy in position 2 (Compound 3); R==H, andR₁=1-methylpiperidin-4-yl-oxy in position 2 (Compound 4); R=H, andR₁=R₂=pyridin-4-yl-oxy in positions 2, and 3 (Compound 5); R=R₂=H, andR₁=1,3-bis-(dimethylamino)-2-propyloxy in position 1 (Compound 6); R=H,and R₁=R₂=3-(piperidin-1yl)propyloxy in positions 1, and 4 (Compound 7);R=H, and R₁=R₂=3-(dimethylamino)phenoxy in positions 2, and 3 (Compound8); R==H, and R₁=pyridin-2-yl-oxy in position 2 (Compound 9); R==H, andR₁=pyridin-3-yl-oxy in position 2 (Compound 10); R=R₂=H, andR₁=3-(dimethylamino)phenoxy in position 1 (Compound 11); R=H, andR₁=R₂=2-(diethylamino)ethylthio in the positions 2, and 3 (Compound 12);R=H, and R₁=R₂=pyridin-3-yl-oxy in positions 2, and 3 (Compound 13);R==H, and R₁=2-(dimethylamino)ethyloxy in position 1 (Compound 14);R==H, and R₁=2-(piperidin-1-yl)ethyloxy in position 1 (Compound 15);R==H, and R₁=2-(piperidin-1-yl)ethyloxy in position 2 (Compound 16);R=R₂=H, and R₁=N-(2-aminoethyl)benzamidoyl-4-oxy trifluoro acetate(Compound 17); R=R₂=H, and R₁=1,3-bis-(trimethylammonium)-2-propyloxydiiodide in position 2 (Compound 18); R=R₂=H, andR₁=3-(trimethylammonium)phenoxy iodide in position 2 (Compound 19);R=R₂=H, and R₁=1,1-dimethylpiperidinium-4-yl-oxy iodide in position 2(Compound 20); R=H, and R₁=R₂=3-(1-methylpiperidinium-1-yl)propyloxyiodide in positions 1, and 4 (Compound 21); R=H, andR₁=R₂=3-(trimethylammonium)phenoxy iodide in positions 2, and 3(Compound 22); R=R₂=H, and R₁=1,3-bis-(trimethylammonium)-2-propyloxydiiodide in position 1 (Compound 23); R=R₂=H, andR₁=3-(trimethylammonium)phenoxy iodide in position 1 (Compound 24); R=H,and R₁=R₂=2-(diethylmethylammonium)ethylthio iodide in positions 2 and3, (Compound 25); and R==H, and R₁=1-methylpyridinium-4-yl-oxy inposition 2 (Compound 26).
 7. The therapeutic method according to claim1, wherein said pharmaceutical composition contains said compound ofgeneral formula (I) incorporated in liposomes or microcapsules.
 8. Thetherapeutic method according to claim 1, wherein said pharmaceuticalcomposition is in the form of solutions for the administration byparenteral route or of preparations for topical administration.
 9. Thetherapeutic method according to claim 8, wherein said preparations fortopical administration are selected from lotions, creams, ointments orgels.
 10. The therapeutic method according to claim 8, wherein saidsolutions are DMSO aqueous solutions, up to
 5000. 11. The therapeuticmethod according to claim 1, further comprising the irradiation of thetissue to be treated with a light source suitable for carrying out thephotodynamic therapy (PDT).
 12. The therapeutic method according toclaim 11, wherein said light source is selected from properly filteredwhite light and laser light having wavelength ranging from 650 to 750nm.